COMPARISON OF TWO CHEMILUMINESCENT AUTOMATED SYSTEMS FOR THE SCREENING of HBsAg, HCV, HIV Colao M. G., T. Capobianco, G. Mazzarelli, F. Parri Serology Laboratory AOU Careggi, Largo Brambilla 3, 50124 Florence, Italy

Background

In the past years, HBsAg, HCV and HIV screening testing was Universal and suitable precautions should be adopted for Aim of this study was to evaluate always required for all patients at the time of hospitalization. all patients regardless the suspect of bearing an infection. the performance and compare This was done mainly as a preventive measure for health care In this context, the clinical laboratory could be active not the results obtained with workers against the biological risk of accidental exposure only as the structure devoted to the testing delivery, but also Chemiluminescent assays for the and as a way to check the status of the people at first hospital as a promoter of the culture of the most appropriateness screening of HBsAg, HCV and HIV admission. of diagnostic requests. At the same time, the availability Ag/Ab, available on two automated Today, in these years of spending review and due to an of diagnostic tests with high level of sensitivity, specificity, platforms with high throughput: increased attention towards the cost-benefit analysis, there robustness and reliability, combined with automation and LIAISON® XL (DiaSorin, Saluggia, is the need to give more attention to the testing requests, traceability of the results to improve the turnaround time of Italy) and Advia Centaur XP (Siemens with a main focus to the patients that really will take an tests execution, can allow to reduce the overall costs, with Healthcare Diagnostics, USA). All advantage from the diagnostic tests, as well as to the use of the reduction of additional tests and investigations, cost of discordant and problematic samples those tests for at-risks patients, and for the screening of the hospitalization and personnel costs. have been tested with additional blood, organs and tissues donors. methods.

MATERIALS AND METHODS

334, 353 and 306 serum samples taken from the daily routine of the serology lab, have been tested with HBsAg, HCV and HIV Ag/Ab respectively. Among them, 43 samples were taken from our serum bank, stored at -20° C and were from problematic patients and /or showing low level of reactivity against at least one marker. The results of these samples were analyzed together with those from the routine. In addition one panel of “rec DNA HBsAg Mutants 2010 ”provided by DiaSorin and the 1st WHO International Reference Panel for B Virus (HBV) Genotypes for Surface Antigen (HBsAg) Assays, PEI code 6100/09, have been evaluated for HBsAg. Tests performed on Advia Centaur XP (Siemens): Tests performed on the LIAISON® XL (DiaSorin): HBsAg II: chemiluminescent enzyme for the qualitative detection of hepatitis B surface antigen HBsAg Quant: chemiluminescent immunoassay for the quantitative determination of HBV surface antigen. (HBsAg). The test utilizes monoclonal to capture anti-HBs. The chemiluminescent reaction is directly The test uses monoclonal antibodies directed against epitopes of the external and highly conserved internal proportional to the amount of HBsAg in the sample. The results are expressed as an Index. Samples with and transmembrane regions of HBsAg with a balanced reactivity against subtypes ad and ay, which ensures a an Index <1 are classified as negative, while samples with an Index ≥ 1 and <50 are positive but require a high sensitivity for the detection of mutants and different genotypes. The instrument automatically calculates confirmatory assay (HBsAg Confirmatory). Samples with an Index ≥ 50 are positive. the concentrations of HBsAg expressed in IU/ml using a stored curve obtained on the basis of dose calibrators HCV: chemiluminescent enzyme immunoassay for the qualitative detection of IgG antibodies to HCV. The test calibrated against the NIBSC standard (WHO code 00/588). The threshold value which distinguishes between employs two recombinant HCV antigens of NS3, NS4, NS5 regions (c200 and NS5) and a synthetic peptide of the presence and absence of HBsAg is 0.05 IU/ml, therefore values <0.05​​ IU/ml are negative; values ≥​​ 0.05 IU/ the core (c22). The chemiluminescent reaction is directly proportional to the amount of anti-HCV present in ml are positive the sample. The results are expressed as an Index. Samples with an index value <0.8 are negative. Samples HCV Ab: chemiluminescent immunoassay for the qualitative determination of specific antibodies against the with an Index value ≥0.8 and <1 are equivocal. Samples with an Index value ≥ 1 are positive. virus of . The solid phase consists of two recombinant antigens (core and NS4) and a third antigen HIV Ag/Ab Combo: chemiluminescent enzyme immunoassay for the simultaneous qualitative detection of (NS3). The presence of antibodies in the samples is determined by comparing the signal of the chemiluminescent HIV p24 antigen and antibodies to HIV type 1, including the group “O”, and / or type 2. The assay employs reaction with the threshold value (cut-off). The analyzer calculates the results: the samples with S / CO <1 are as a solid-phase recombinant antigens of the HIV-1 ( / gp120), a protein of the ENV HIV-2 negative; Samples with S / CO ≥ 1 are positive. (gp36), a synthetic peptide for the detection of HIV group “O” antibodies and two monoclonal antibodies HIV Ag/Ab: chemiluminescent immunoassay for the qualitative detection of HIV-1 combined p24 antigen and specific to detect the HIV p24 antigen. The chemiluminescent reaction is directly proportional to the amount antibodies to HIV-1 (group M and group O) and / or HIV-2. The test uses two different integrals of reagents, of HIV and / or p24 antigen present in the sample. The results are expressed as an Index. Samples one for the detection of antibodies to HIV and one for the detection of p24 antigen, and uses recombinant with an Index value <1 are negative. Samples with an Index value ≥ 1 are positive. antigens gp41 of HIV-1 (group M and group O) and gp35 of HIV-2. The presence of p24 antigen and / or of anti- HIV antibodies is determined by comparing the signal of the chemiluminescent reaction with the threshold value (cut-off) as provided by each specific assay. The analyzer calculates the combined results: Samples with S / CO <1 are negative; Samples with S / CO ≥ 1 are positive. Additional tests for discordant results and “problematic” samples with weak reactivity: • Abbott Architect HBsAg quantitative: chemiluminescent immunoassay performed on i2000SR Architect for the quantitative detection of HBV surface antigen. Values <0.05​​ IU/ml are negative; values ≥ 0.05 IU/ml are positive. • Abbott Architect HBsAg qualitative II: chemiluminescent immunoassay for the detection of HBV surface antigen, values <1​​ S/CO are negative; values ≥​​ 1 S/CO are positive. • Abbott Architect anti-HCV: chemiluminescent immunoassay for the qualitative detection of antibodies to structural and non-structural of HCV (core, NS3 and NS4). Values <1​​ S/CO are negative; values ≥​​ 1 S/CO are positive. • INNO-LIATM HCV Score Innogenetics (now Fujirebio): 3rd generation Immunoblot for the evaluation of antibodies against antigens of structural and non-structural HCV regions (core, E2, NS3, NS4A, NS4B, NS5A). For the interpretation of the results the software LiRASTM have been used according to the criteria proposed by Innogenetics. • Abbott Architect HIV Ag/Ab Combo: chemiluminescent immunoassay for the simultaneously qualitative detection of HIV p24 antigen and antibodies against recombinant antigens and synthetic peptides from sequences of the viral transmembrane protein of HIV-1 and 2. Values ​​<1 S/CO are negative; values ​​≥ 1 S/CO are positive. • Siemens Advia Centaur XP HIV Ab 3rd generation: chemiluminescent enzyme immunoassay for the qualitative detection of antibodies to HIV (type 1, type 2 and subtype O) directed against recombinant antigens of the ENV and the core. Values ​​<1 index are negative; values ≥ 1 index are positive. • bioMérieux VIDAS HIV p24 II: quantitative test for HIV p24 antigen detection using ELFA technology (Enzyme Linked Fluorescent Assay). Values ​​<3 pg/ml are negative; values ​​≥ 3 and <5 pg/ml are equivocal; values ​​≥ 5 pg/ml are positive. RESULTS

The correlation between the tests performed on LIAISON® XL and Advia Centaur XP was Diagnostic sensitivity of Advia Centaur XP HCV was 94.7%, and diagnostic specificity was 100% for HBsAg, 97.4% for anti HCV and 97.7% for HIV Ag / Ab (see Tables 1, 2, 3). 98.7%. The 7 discordant results for HIV Ag/Ab (3 positive with LIAISON® XL and 4 positive Diagnostic sensitivity of LIAISON® XL HBsAg Quant was 100%, and diagnostic specificity with Advia Centaur XP) showed a weak reactivity with both systems (see Table 5): all were was 100%. Diagnostic sensitivity of Advia Centaur XP HBsAg was 100%, and diagnostic negative with the 4th generation test performed on Architect for the evaluation of Ag/Ab, specificity was 100%. with the 3rd generation test run on Centaur for the evaluation of anti-HIV antibodies only, The 9 discordant results for HCV (5 positive with LIAISON® XL and 4 positive with Advia and with the test for the HIV p24 antigen alone performed on Vidas. Centaur XP) all showed a weak positive results (see Table 4). The test run on Architect Diagnostic sensitivity of LIAISON® XL HIV Ab/Ag was 100%, and diagnostic specificity was agreed with the positivity of a single positive sample with LIAISON® XL. Out of 5 samples 99%. positive with LIAISON® XL and negative with Advia Centaur XP, the INNO-LIA confirmed the Diagnostic sensitivity of Advia Centaur XP HIV Ag/Ab was 100%, and diagnostic specificity positivity of 2 samples (true positive for LIAISON® XL and false negative with Advia Centaur was 98.6%. XP), while the 3 other samples were classified as negative (false positive with LIAISON® XL). The sensitivity of the HBsAg Quantitative test on LIAISON® XL on the panel of mutants was The INNO-LIA did not confirm the 4 samples positive with Advia Centaur XP and negative excellent (see Table 6). with LIAISON® XL (false positive with Advia Centaur XP). The molecular biology test of Both HBsAg assays showed a good sensitivity across the different genotypes, when using the Real Time PCR, performed on the 3 samples available, gave a result below the sensitivity 1st WHO International Reference Panel for (HBV) Genotypes for Hepatitis threshold of the method used (<15 IU/ml). Diagnostic sensitivity of LIAISON® XL HCV was B Surface Antigen (HBsAg) Assays, PEI code 6100/09 (see Table 7). 100%, and diagnostic specificity was 99%.

Table 1 Table 2 Table 3 Table 4

® Roche Real time HBsAg ADVIA CENTAUR XP HCV Ab ADVIA CENTAUR XP HIV Ag/Ab ADVIA CENTAUR XP Sample LIAISON XL Advia Centaur Architect Innogenetics PCR ID HCV S/CO XP HCV Index HCV S/CO Immunoblot HCV Score HCV RNA LIAISON® XL neg pos tot LIAISON® XL neg pos tot LIAISON® XL neg pos tot 31s 1.60 Neg 4.62 Pos Tbl 1.40 Neg Neg Pos 9605 1.00 Neg Neg Neg neg 321 0 321 neg 310 4 314 neg 292 4 296 Psr 1.60 Neg Neg Neg R001 1.20 Neg Neg Neg < 15 IU/mL pos 0 13 13 pos 5 34 39 pos 3 7 10 18s Neg 1.26 Neg Neg 23s Neg 1.10 Neg Neg tot 321 13 334 tot 315 38 353 tot 295 11 306 R002 Neg 1.34 Neg Neg < 15 IU/mL R004 Neg 3.60 Neg Neg < 15 IU/mL

Table 5 Table 6 Table 7

® ® ® Advia Siemens LIAISON LIAISON LIAISON Advia Architect DiaSorin Abbott Abbott Sample ID LIAISON® XL Siemens Centaur XP BioMerieux Sample LIAISON® XL Architect Architect Advia HBV HBV Sample XL HIV XL HIV XL HIV Centaur XP HIV Ab HIV Ag/ Vidas HIV Mutation Centaur panel PEI Origin Murex HBsAg Advia ID Ab Ag Ab/Ag HIV Ag/Ab Ab ID HBsAg HBsAg HBsAg II 6100/09 Sub-Genotype Sub-Genotype Quant HBsAg II (3° gen) p24 pg/mL Quant Quant Qualitative XP HBsAg S/CO S/CO S/CO Index Index S/CO Qualitative Prl 4.14 Neg Pos Neg Neg Neg Neg CO = CO = CO = CO = 1 A1 adw2 South Africa REACTIVE REACTIVE Sen 1.19 Neg Pos Neg Neg Neg Neg 0.05 IU/mL 0.05 IU/mL 1.00 Index 1.00 Index 2 A1 adw2 Brazil REACTIVE REACTIVE 9354 Neg 3.48 Pos Neg Neg Neg Neg 1 T123N 0.92 0.05 19.98 10.18 3 A2 adw2 Germany REACTIVE REACTIVE Gls Neg Neg Neg 1.63 Neg Neg Neg 2 T123N-T124S 0.19 0.02 9.94 1.06 4 B1 adw2 Japan REACTIVE REACTIVE 2 Neg Neg Neg 1.20 Neg Neg Neg 3 P142L-F/Y143H-D144E-G145R 0.26 0.02 < 0.10 0.90 5 B2 adw2 Japan REACTIVE REACTIVE 5 Neg Neg Neg 1.69 Neg Neg Neg 4 I110R-S117I-G119R-T123N 0.69 0.04 < 0.10 2.67 6 C2 adr Japan REACTIVE REACTIVE 7 Neg Neg Neg 1.40 Neg Neg Neg 5 122+DT 1 0.06 42.08 3.58 7 C2 adr Japan REACTIVE REACTIVE 6 122+DT-G145R 1.4 0.06 < 0.10 4.46 8 C2 adr Russia REACTIVE REACTIVE 7 G145R 0.39 0.23 5.57 9.13 9 D1 ayw2 Germany REACTIVE REACTIVE 8 D144A 0.56 0.33 24.01 41.33 10 D2 ayw3 Russia REACTIVE REACTIVE 9 P142L- G145R 0.49 0.19 7.41 7.96 11 D3 ayw2 South Africa REACTIVE REACTIVE 10 P142S- G145R 0.55 0.22 9.5 9.35 12 E ayw4 West Africa REACTIVE REACTIVE 13 F2 adw4 Brazil REACTIVE REACTIVE Negative 14 F2 adw4 Brazil REACTIVE REACTIVE matrix < 0.030 0.02 < 0.10 0.19 15 H adw4 Germany REACTIVE REACTIVE

Conclusions Both systems (LIAISON® XL and Advia Centaur XP) demonstrated to have available excellent assays for the evaluation of HBsAg, HCV Ab and HIV Ag/Ab, suitable for routine use and characterized by very high diagnostic sensitivity and specificity. Excellent results were obtained on samples of the panel of mutants “rec HBsAg DNA Mutants 2010”, with the HBsAg Quant DiaSorin that has correctly identified all the mutations. Both assays were able to detect the different genotypes of the PEI panel. Poster P06.01 / 5 - 17th Annual Meeting of the European Society for Clinical Virology/ESCV - September 28 – October 1, 2014 - Prague